[논문]프로안토시아니딘의 항염증효과

천혜진; 박선영; 장희지; 조다영; 정지원; 박기민; 정경미; 김진경

doi:10.5352/jls.2019.29.4.484

프로안토시아니딘의 항염증효과
Proanthocyanidins Suppresses Lipopolysaccharide-stimulated Inflammatory Responses via Heme Oxygenase-1 Induction in RAW264.7 Macrophages 원문보기

생명과학회지 = Journal of life science, v.29 no.4 = no.228, 2019년, pp.484 - 491

천혜진 (대구가톨릭대학교 의생명과학과) , 박선영 (대구가톨릭대학교 의생명과학과) , 장희지 (대구가톨릭대학교 의생명과학과) , 조다영 (대구가톨릭대학교 의생명과학과) , 정지원 (영천여자고등학교) , 박기민 (영천여자고등학교) , 정경미 (영천여자고등학교) , 김진경 (대구가톨릭대학교 의생명과학과)

초록
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프로안토시아니딘(proanthocyanidins)은 식물계에 가장 풍부한 폴리페놀성 화합물로 다양한 고등식물의 뿌리, 잎, 열매, 나무껍질 등에 널리 존재할 뿐만 아니라 이러한 원료로 만들어진 차, 와인, 맥주 등과 같은 식품에도 상당량 함유되어 있다. 세포 및 실험동물을 이용한 다수의 연구보고에 의하면 프로안토시아니딘은 항산화활성 및 면역조절활성, DNA 복구 및 항종양 작용과 같은 인체 건강에 유익한 무수한 효과를 가지고 있는 것으로 밝혀졌다. 면역 세포 중 대식세포(macrophage)는 염증반응을 매개하는 중요한 세포로 외부 병원체 제거에 중요한 역할을 수행하고 있다. 그러나 대식세포가 만성 염증을 유발하고 비만, 당뇨병, 대사 증후군 및 암과 같은 다양한 질병에 관여한다는 것 또한 널리 보고되어왔다. 본 연구에서는 마우스의 대식세포주인 RAW264.7세포를 이용하여 프로안토시아니딘의 항염증활성의 일단이 Heme oxygenase-1 (HO-1)의 유도에 의해서 매개됨을 밝혔다. RAW264.7세포에 프로안토시아니딘을 처리한 결과 세포독성을 보이지 않은 농도에서 HO-1의 발현을 증강시켰다. 또한 프로안토시아니딘의 처리는 HO-1의 발현을 조절하는 핵심 전사인자인 Nrf (nuclear factor-erythroid 2-related factor)-2의 핵으로의 이동을 유의적으로 증가시켰다. 프로안토시아닌딘의 처리는 LPS (lipopolysaccharide)에 의해 유도된 NO (nitric oxide)의 생성 및 iNOS (inducible NO synthase)의 발현과 염증성 사이토카인의 생성 및 발현도 유의적으로 억제 하였다. 이러한 결과는 프로안토시아니딘의 항염증제제로서의 개발 가능성을 제시하는 결과이다.

Abstract ▼ AI-Helper

Proanthocyanidins are naturally occurring polyphenolic compounds abundant in many vegetables, plant skins (rind/bark), seeds, flowers, fruits, and nuts. Numerous in vitro and in vivo studies have demonstrated myriad effects potentially beneficial to human health, such as antioxidation, immunomodulation, DNA repair, and antitumor activity. Among immune cells, macrophages are crucial players in a variety of inflammatory responses to environmental conditions. However, it has been widely reported that macrophages cause chronic inflammation and are involved in a variety of diseases, such as obesity, diabetes, metabolic syndrome, and cancer. In this study, we report the suppressive effect of proanthocyanidins via the heme oxygenase-1 (HO-1)-related system, on the immune response of the LPS-stimulated mouse macrophage cell line RAW264.7. Increased HO-1 expression at mRNA and protein levels were found in proanthocyanidins-treated RAW264.7 cells. Further, proanthocyanidins enhanced nuclear factor-erythroid 2-related factor 2 translocation into the nucleus. RAW264.7 cells were treated with lipopolysaccharide (LPS) with or without proanthocyanidins, and inflammatory mediator expression levels were assessed. Proanthocyanidins treatment resulted in the attenuation of nitric oxide production and inducible nitric oxide synthase expression in LPS-stimulated RAW264.7 cells. In addition, mRNA and protein expression of proinflammatory cytokines, such as tumor necrosis factor-${\alpha}$ and interleukin-6, was inhibited by proanthocyanidins treatment in LPS-stimulated RAW264.7 cells. These findings support proanthocyanidins as a promising anti-inflammatory agent.

주제어

표/그림 (4)

그림 Fig. 1. Effect of proanthocyanidins on cell viability in RAW264.7 macrophages.
그림 Fig. 2. Proanthocyanidins induced HO-1 protein expression.
그림 Fig. 3. Proanthocyanidins significantly reduced LPS-induced NO production and iNOS expression.
그림 Fig. 4. Proanthocyanidins treatment shown significant inhibitory effect on proinflammatory cytokines.

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문제 정의

A number of pharmacological effects have been reported for proanthocyanidins, such as antiviral, antimicrobial, anti-HIV, anti-oxidative and antitumor- promoting properties, as well as cardiotonic and anti- arteriosclerotic activities [1, 11, 16, 19, 22]. In this study, we examined the effects of proanthocyanidins on lipopolysaccharide (LPS)-induced inflammatory responses in murine RAW264.7 macrophages. LPS is a component of the outer membrane of gram-negative bacteria.

제안 방법

In this study, we evaluated the functions of proanthocyanidins- induced expression of HO-1 by macrophage cells under LPS treatment. First, we showed that proanthocyanidins addition induces HO-1 mRNA and protein expression without obvious cell damage.

대상 데이터

PRO-PREP^™ Protein Extraction Solution and NE-PER^™ Nuclear and Cytoplasmic Extraction Reagents were obtained from iNtRON Biotechnology(Seongnam, Korea) and Thermo Scientific^™(Waltham, MA, USA), respectively. Antibodies against inducible nitric oxide synthase (iNOS) and HO-1 were purchased from BD Biosciences (San Jose, CA) and Santa Cruz Biotechnology(Santa Cruz, CA), respectively.
The primers used in this study corresponded to mouse iNOS: F 5'-CCT CCT CCA CCC TAC CAA GT-3', R 5'-CAC CCA AAG TGC TTC AGT CA-3', mouse IL-6: F 5'-CAT CCA GTT GCC TTC TTG GGA-3', R 5'-CTG AAG GAC TCT GGC TTG TC-3', mouse TNF-α: F 5'-TGT CTC AGC CTC TTC TCA TT-3' R 5'-AGA TGA TCT GAG TGT GAG GG-3', mouse HO-1: F 5'-GCT TGT TGC GCT CAT TCT CC-3’ R 5'- GCC ACC AAG GAG GTA CAC ATA -3' and mouse GAPDH: F 5'-TCT TGC TCA GTG TCC TTG C-3'.

데이터처리

The data were presented means ± SEM. The values were evaluated by one-way ANOVA with Bonferroni multiple comparison range tests using GraphPad Prism 4.0 software(GraphPad Software Inc, San Diego, CA). Statistically significant differences were considered at a P-value < 0.

이론/모형

DNA was eliminated from total RNA using RNA Qualified RNase-Free DNase (Promega) and cDNA was synthesized by GoScript^™ Reverse Transcription System (Promega). qRT-PCR assay was carried out with LightCycler (Roche Diagnostics, Basel, Switzerland) using LightCycler FastStart DNA Master SYBR Green I (Roche Diagnostics). All the experiments were repeated twice in triplicate each time.

성능/효과

3B). Our results also showed that proanthocyanidins significantly reduced the expression of iNOS gene induced by LPS (Fig. 3C). These data demonstrate proanthocyanidins can restrain the production of NO by down-regulating the expression of iNOS.

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